research day - University of Toronto Department of Obstetrics and ...

31
ABSTRACT #O6
MECHANISM OF FACTOR INHIBITING HIF-1 (FIH-1) REGULATION IN
PLACENTAE FROM NORMAL AND HELLP SYNDROME PREGNANCIES
Antonella Racano[G](1, 2), Alessandro Rolfo (1), and Isabella Caniggia (1,2,3).
(1) Samuel Lunenfeld Research Institute, Mount Sinai Hospital; Departments of (2) Physiology
and (3) Obstetrics and Gynecology, Faculty of Medicine, University of Toronto.
Objective: Factor Inhibiting HIF1 (FIH) is an O 2 -dependent asparaginyl hydroxylase that
represses the transcriptional activity of Hypoxia Inducible Factor 1 (HIF1), the key factor that
mediates the cellular response to hypoxia. Albeit FIH is a key regulator of the hypoxic response,
little is known about its regulation. Recent studies conducted in cells have shown that Seven In
Absentia Homologue 1 (Siah1), an O 2 sensor and E3 ubiquitin ligase, facilitates FIH degradation
via the ubiquitin-proteasome pathway. We have previously reported that HIF1 plays an important
role in placental development and that HIF1 expression is increased in preeclampsia (PE), resulting
in enhanced HIF1 transcriptional activity. Herein, we investigated the expression of FIH in normal
and pathological placentae and examined the contribution of Siah1 in regulating its stability.
Methods: First trimester (n=30), HELLP (n=14), and age-matched control (n=20) placentae were
used. FIH and Siah1 protein levels were assessed by Western blot (WB). FIH/Siah1 co-localization
was examined by immunofluorescence (IF) in JEG3 cells. To test FIH-Siah1 association and FIH
degradation, FIH immunoprecipitation (IP) was performed followed by Siah1 and Ubiquitin WB,
respectively, in human placental lysates.
Results: During placental development, FIH exhibited a peak of expression between 10-12 weeks
of gestation, at a time when Siah1 levels were low. Preliminary IP studies indicate that during
placental development FIH associates with Siah1 and that this association is increased with
advancing gestation. IF staining in JEG3 cells suggests a positive perinuclear co-localization of
both FIH and Siah1. Of clinical relevance, FIH expression was significantly decreased in HELLP
syndrome relative to preterm and term controls. Moreover, IP studies indicated that FIH/Siah1
association and FIH ubiquitination are increased in HELLP placentae relative to controls,
suggesting increased FIH degradation in placentae from pregnancies complicated by HELLP
syndrome.
Conclusion: Our data highlights a novel role for Siah1 as a potential regulator of FIH stability in
the developing placenta. Therefore, decreased FIH expression in placentae from HELLP
pregnancies may be due to its degradation via Siah1.
Funded by: CIHR/IGH and Genesis Research Foundation